Abstract: The objective of this proposal is to better understand the properties of Myo3B and its role as a transporter in actin bundle based structures such as the stereocilia of inner ear hair cells. Class III myosins are known actin based motors and are unique because they contain an N-terminal kinase domain that serves to autoregulate motor activity. In vertebrates there exists two class III myosin isoforms; Myo3A and Myo3B. Class III myosins are expressed in inner ear hair cells, photoreceptors, brain, testis, and intestines. Both Myo3A and Myo3B have been shown to bind the actin bundling protein Espin and transport it to the tips of actin protrusions to promote elongation. Myo3A harbors deafness associated mutations while Myo3B is proposed to partially compensate for lack of functional Myo3A, which results in delayed onset deafness. In addition, we recently discovered that Myo3B is strongly regulated by physiological concentrations of calcium, while Myo3A is not. Therefore, it is crucial to characterize the intrinsic motor properties of the Myo3B motor and understand how it is calcium regulated to determine its function in parallel actin based structures. We will investigate the calcium regulation of Myo3B with in vitro assays such as steady-state ATPase, in vitro motility, and transient kinetic analysis with purified proteins which will allow us to determine the intrinsic motor properties of MYO3B. In addition, cell biological studies using cultured cells will enable us to examine the calcium regulation of Myo3B localization and impact on actin protrusion dynamics. Overall, this study will provide fundamental information about the motor mechanism of Myo3B, which will reveal crucial information about its function in the stereocilia of inner ear hair cells.